Endogenous pancreatic progenitor cells represent a potentially important resource for cell replacement therapy in diabetes, and may also act as cells of origin for pancreatic cancer. However, the presence, location and identity of multipotent progenitor cells in adult vertebrate pancreas remain unknown. Among the various pancreatic cell types proposed as candidate progenitors, centroacinar and terminal duct cells remain perhaps the most poorly characterized. We have developed a novel strategy to both visualize and isolate a low abundance subset of these cells from adult mouse and zebra fish pancreas, and generated exciting preliminary data suggesting that these cells carry a multipotent progenitor capacity. This strategy involves isolating cells based upon high expression of the retinoic acid-synthesizing enzyme, Aldehyde Dehydrogenase 1 (ALDH1). During development, ALDH1-expressing cells are located in the "tips" of the branching pancreatic epithelium, a location recently associated with progenitor activity. In adult pancreas, they are frequently observed in centroacinar and terminal duct locations. When isolated by FACS and analyzed by RT-PCR, ALDH1-expressing centroacinar and terminal duct cells showed no expression of differentiated lineage markers, but did show enrichment of transcripts for both c-Met and SDF1, markers previously associated with progenitor cells in pancreas and other tissues. When placed into suspension culture, ALDH1-expressing cells are uniquely able to form "pancreatospheres", and over time these spheres generate functional endocrine and exocrine cells. In addition, the spheres maintain a subpopulation of cells expressing ALDH1, Hes1 and Sox9, suggesting a capacity for self-renewal. When ALDH1-expressing cells are isolated from adult mouse pancreas and injected into micro-dissected E12.5 pancreatic dorsal buds, they are uniquely able to contribute to the emerging endocrine and exocrine lineages. Based on these exciting studies, we hypothesize that ALDH1-expressing pancreatic epithelial cells may function as multipotential progenitors in both in mouse and zebra fish pancreas, and also serve as important local signaling centers by virtue of their ability to synthesize retinoic acid. We plan to test these hypotheses through the application of rigorous in vivo lineage tracing techniques, as well as the independent ablation of both ALDH1 enzymatic activity and ALDH1-expressing cells, using both mouse and zebra fish model systems. These studies will set the stage for the eventual expansion and direct manipulation of these cells, potentially providing a novel pancreatic progenitor population that can be harnessed to generate new b-cells in the context of either endogenous regeneration and/or exogenous replacement therapy. PUBLIC HEALTH RELEVANCE: Pancreatic progenitor cells represent a potentially important resource for cell replacement therapy in diabetes. This project will characterize a novel population of progenitor cells in mouse and zebra fish pancreas, in hopes that they will provide a renewable resource for replacement b-cells.